Mullerian Inhibiting Substance (MIS), a gonadal glycoprotein of the TGF- beta family, has developmental, tissue, and sex specific expression, characterized by constitutive expression in the Sertoli cells of male fetal and postnatal testes, and delayed expression in the granulosa cells of developing follicles of the adolescent and the adult ovary. Its dramatic morphological effects on the regressing Mullerian duct of the male embryo, its meiotic inhibitory effect on immature oocytes, and the antiproliferative effect of MIS against cell lines derived from tumors of Mullerian duct origin, i.e., endometrial or ovarian tumors, in addition to its G1 cell cycle arrest effect, also characteristic of other members of the TGF-beta family, make it likely that MIS is a tumor suppressor and that MIS or one of its congeners might be used as a therapeutic agent against tumors of Mullerian Duct origin in the clinical setting. At the least, its structure and function can serve as a paradigm for the entire family and can provide the basis for rational novel drug design. Human MIS has been cloned, and recombinant protein (rhMIS) expressed; recently after a decade of repeated effort, the ligand has been successfully labelled so that it retains biological activity. This important tool can now be used to identify the MIS receptor. Expression libraries from cells and tissues responsive to MIS have been constructed. In this grant we propose to use the bioactive biotinylated MIS to confirm which of the candidate type I (R1, R2, R3, R4) and type II receptors already cloned by this laboratory and others are the authentic MIS receptors, or to further screen these libraries to clone the true MIS receptor, using flow cytometry and Fluorescent Activated Cell Sorting (FACS) to select positive clones. Once identified the receptor probes can be used to define the range of MIS targets and to determine the percentage of the human ovarian cancer patients that are MIS type II receptor positive, and the percentage of receptor positive tumors that are MIS respo ive. Parallel efforts will be made to enhance expression of MIS and its variants by l) optimizing secretion of rhMIS from Chinese Hamster Ovary (CHO) cells transfected with MIS genomic DNA, 2) producing secreted MIS from a Pichia pastoris yeast expression system, 3) amplifying expression of cleavable MIS (MIS Arg/Arg) from CHO cells, and 4) expressing soluble MIS C in a thioreductase deficient E. coli system. The MIS so produced will be used for in vitro and in vivo antiproliferative studies of MIS, crystallography of MIS and/or MIS C and cocrystallography of MIS C with the extracellular domain of the MIS type II receptor, and, if necessary, affinity purification of receptor. Patients with early premenopausal, familial ovarian cancer will be studied for genetic defects in MIS or the MIS receptor. Thus, with the true MIS receptor complex in hand, we can preselect patients for treatment and test the responsiveness of receptor positive tumors to MIS. In preparation for Phase I clinical trials, we will then select or further design the best congener or construct of MIS, or a more downstream or upstream product to develop as therapeutics against the lethal carcinomas of Mullerian duct origin.